PROJECT SUMMARY AND ABSTRACT Patent foramen ovale (PFO), a residual tunnel between the right and left atria, is associated with more than 150,000 strokes per year in the US alone. PFOs facilitate paradoxical embolism by allowing venous clots to travel directly to the brain. But only a small portion (10-17%) of PFO stroke patients have a known tendency to form venous clots: we propose to explore PFO physiology as a contributor to prothrombotic condition in the 80- 90% of patients without known hypercoagulable state. Since PFOs are common in the general population (25- 30% of adults), and recent clinical trials excluded 80% high risk individuals, there is a dire need to understand the mechanism of PFO stroke better to individualize treatment. The original ESI R01 project hypothesized that the PFO physiology may create a procoagulable condition. And with a combined translational approach we examined intra-atrial blood before and after endovascular PFO closure and have identified circulating factors indicative of the degree of PFO-related shunting, an important recurrent risk marker. In this renewal, we aim to: 1) Construct and validate a panel of biomarkers to measure the PFO-related shunting state from baseline venous blood. The panel will be tested in a prospectively enrolled cohort of PFO stroke patients, and compared to shunting as reported by echocardiogram, with non-PFO stroke controls. 2) Assess the effect of novel PFO-related clinical risk factors in predicting stroke recurrence, both independently and in combination with the biomarker panel. 3) Explore the functional effect of homocysteine (Hcy) on human brain endothelium in cell culture. Hcy is the marker most affected by PFO closure, and has been implicated in white matter disease and blood-brain-barrier injury. It is hypothesized to participate in multiple pathways relevant to the PFO shunting circulatory state which this study aims to explore. In this resubmission for the first R01 renewal, we are deeply grateful for the reviewers? overall enthusiasm and constructive comments. We have revised this resubmission extensively in response to all the reviewers? critiques including: 1) addition of new detailed statistical analysis and power analysis for each aim; 2) addition of new senior biostatistician; 3) providing new data as requested; 4) re-designing the study to avoid selection bias with plan for multi-center collaboration; 5) including new non-PFO stroke control arm; 6) providing feasibility of recruitment and methods in new publications. Ultimately, the project aims to investigate mechanisms of PFO physiology and validate markers that individualize clinical decision-making with novel PFO-specific risk factors, by providing clinicians with practical means, such as a blood test, to assess individual patients? risk of PFO-related stroke or neurovascular injury.